G protein-coupled receptors (GPCRs) represent the largest family of signal-transducing molecules known. For example, GPCRs comprise more than 4% of the genes in Caenorhabditis elegans. GPCRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of GPCRs has been found in a growing number of human diseases and GPCRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how GPCRs function at the molecular level is an important goal of biological research. We have used receptors for thyrotropin-releasing hormone (TRH) (TRH-Rs) as model GPCRs to study their structure and function. During this year, we have studied several new aspects of TRH-R structure and function. We compared several aspects of the biology of the two mouse TRH receptor types 1 (mTRH-R1) and 2 (mTRH-R2), which are 50% identical at the amino acid level. We studied the different signaling pathways/G proteins that are activated by mTRH-R1 versus mTRH-R2. We also continued our study of a viral GPCR, Kaposi's sarcoma-associated herpesvirus GPCR, and began studies of the receptor for thyrotropin (thyroid-stimulating hormone, TSH). In another aspect of the project, we synthesized several novel TRH analogs in which the histidine residue was modified to conain bulky alkyl groups and studied their biological properties. These analogs provided new insights into the size of the binding pocket for TRH within TRH-R1. Lastly, we have been exploring differences in the binding of mTRH-R1 and mTRH-R2 for competitive, small organic molecule, inverse agonists. We have tentatively identified a new series of 1-(phenyl)isoquinoline carboxamide analogs that discriminate between mTRH-R1 and mTRH-R2.